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Team:Warsaw/Calendar-Main/14 August 2009
From 2009.igem.org
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| - | <h3>Cloning Bax into | + | <h3>Cloning Bax into pSB1A3 plasmid</h3> |
<h4>Justyna</h4> | <h4>Justyna</h4> | ||
<p>Task 1:</p> | <p>Task 1:</p> | ||
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<p>Task 2:</p> | <p>Task 2:</p> | ||
<ul> | <ul> | ||
| - | <li>Digestion and gel-out of | + | <li>Digestion and gel-out of pSB1A3 plasmid</li> |
</ul> | </ul> | ||
<br/> | <br/> | ||
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<p>Task 3:</p> | <p>Task 3:</p> | ||
<ul> | <ul> | ||
| - | <li>Ligation of Bax and | + | <li>Ligation of Bax and pSB1A3</li></ul> |
<p>Methods:</p><ul> | <p>Methods:</p><ul> | ||
<li>Ligation mix:</li> | <li>Ligation mix:</li> | ||
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2.5 μl - Tango buffer (Fermentas, 10x) | 2.5 μl - Tango buffer (Fermentas, 10x) | ||
2.0 μl - dNTPs (EURx, 5μlM) | 2.0 μl - dNTPs (EURx, 5μlM) | ||
| - | 5.0 μl - digested | + | 5.0 μl - digested pSB1A3 (~100 ng) |
14.5 μl - Bax (~ 105 ng) | 14.5 μl - Bax (~ 105 ng) | ||
in 25.0 μll | in 25.0 μll | ||
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<p>Task 4:</p> | <p>Task 4:</p> | ||
<ul> | <ul> | ||
| - | <li>Insertion of Bax- | + | <li>Insertion of Bax-pSB1A3 plasmid into bacteria</li></ul> |
<p>Methods:</p><ul> | <p>Methods:</p><ul> | ||
<li>Previously prepared chemicompetent bacteria were used.</li> | <li>Previously prepared chemicompetent bacteria were used.</li> | ||
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<ul><li> Isolate the plasmid prepared in <a href="https://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/13_August_2009">13.08.09</a></ul></li> | <ul><li> Isolate the plasmid prepared in <a href="https://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/13_August_2009">13.08.09</a></ul></li> | ||
<p>Methods:</p><ul> | <p>Methods:</p><ul> | ||
| - | <li>Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf" | + | <li>Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf">here</a></li></ul> |
<br/> | <br/> | ||
<p>Task 2:</p><ul><li>Restriction digest of following sequences from the <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid: | <p>Task 2:</p><ul><li>Restriction digest of following sequences from the <a href="http://partsregistry.org/Part:pSB1A3"><span style="color: black">pSB1A3</a></span> plasmid: | ||
| Line 85: | Line 85: | ||
<p>Methods:<p/> | <p>Methods:<p/> | ||
<ul> | <ul> | ||
| - | <li>Fragments of agarose gel were carefully cut out and subsequently frozen in -20°C./ | + | <li>Fragments of agarose gel were carefully cut out and subsequently frozen in -20°C></li></ul> |
| + | <p>Results</p> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2009/9/9a/P53_R0080_E0022_C0040_B0032_digest_14_08_09.png" width="40%" height="40%"></center> | ||
| + | <font face="Times New Roman" size="3"><p><div style="text-align: center;">Verification of the digestions</div></p></font> | ||
<p><b>Comment:</b></p> | <p><b>Comment:</b></p> | ||
<p>Restriction digests of samples containing <a href="http://partsregistry.org/Part:BBa_E0022"><span style="color: black">BBa_E0022</a></span> and <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></span></li> were unsuccessful, probably the probes were contaminared. Because of this fact I decided to repeat the digest. <p><br/> | <p>Restriction digests of samples containing <a href="http://partsregistry.org/Part:BBa_E0022"><span style="color: black">BBa_E0022</a></span> and <a href="http://partsregistry.org/Part:BBa_B0032"><span style="color: black">BBa_B0032</a></span>+<a href="http://partsregistry.org/Part:BBa_C0040"><span style="color: black">BBa_C0040</a></span></li> were unsuccessful, probably the probes were contaminared. Because of this fact I decided to repeat the digest. <p><br/> | ||
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<p>Task 6:</p><ul><li>Prepare chemocompetent TOP10 bacteria strain (I worked with Kuba)</ul></li> | <p>Task 6:</p><ul><li>Prepare chemocompetent TOP10 bacteria strain (I worked with Kuba)</ul></li> | ||
<p>Methods:<p/><ul> | <p>Methods:<p/><ul> | ||
| - | <li>Detailed protocol is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/22_July_2009">here</a> | + | <li>Detailed protocol is described <a href="https://2009.igem.org/Team:Warsaw/Calendar-Main/22_July_2009">here</a></li> |
Latest revision as of 15:48, 28 September 2009
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Cloning Bax into pSB1A3 plasmid
Justyna
Task 1:
- Gel-out Bax PCR product
Methods:
- Bax PCR products were isolated from agarose gel using A&A Gel-Out kit.
Task 2:
- Digestion and gel-out of pSB1A3 plasmid
Methods:
- Digestion of isolated plasmids by XbaI and SpeI
- Reaction mixture composition: 10.0 μl purified plasmid DNA product, 0.5 μl XbaI (Fermentas),0.5 μl SpeI (Fermentas), 2 μl Buffer Orange (Fermentas), 7.0 μl MQ water
- The digestion was performed overnight and it was subsequently inactivated via heating in 80°C for 20 minutes.
- In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids and isolated using A&A Gel-Out kit
Task 3:
- Ligation of Bax and pSB1A3
Methods:
- Ligation mix:
1.0 μl - T4 ligase 2.5 μl - Tango buffer (Fermentas, 10x) 2.0 μl - dNTPs (EURx, 5μlM) 5.0 μl - digested pSB1A3 (~100 ng) 14.5 μl - Bax (~ 105 ng) in 25.0 μll
Task 4:
- Insertion of Bax-pSB1A3 plasmid into bacteria
Methods:
- Previously prepared chemicompetent bacteria were used.
Assembly of endosomal detection operon
Marcin
Task 1:
- Isolate the plasmid prepared in 13.08.09
Methods:
- Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
Task 2:
- Restriction digest of following sequences from the pSB1A3 plasmid:
Methods:
- Reaction mixture composition:
20 μl purified plasmid DNA product 1 μl XbaI (Fermentas) or 0,5 μl SpeI (Fermentas) in the case of R0080 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 24 μl MQ water
Task 3:
- Gel-out of digest sequences described in Task 1
Methods:
- Fragments of agarose gel were carefully cut out and subsequently frozen in -20°C>
Results
Verification of the digestions
Comment:
Restriction digests of samples containing BBa_E0022 and BBa_B0032+BBa_C0040 were unsuccessful, probably the probes were contaminared. Because of this fact I decided to repeat the digest.
Task 4:
- Another digest of E0022 and C0040+B0032
Methods:
- Reaction mixture composition:
20 μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 24 μl MQ water
Task 5:
- Transformation of chemocompetent E. coli strain DH5&alpha
Constructs to transform:
Methods:
- Ligation prepared in 08.08.09 was stopped via thermal inactivation in 80°C for 20 minutes
- Detailed protocol of transformation is described here.
Task 6:
- Prepare chemocompetent TOP10 bacteria strain (I worked with Kuba)
Methods:
- Detailed protocol is described here
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